Abstract

The effect of the dopamine autoreceptor antagonist (−)DS121 on wakefulness, locomotor activity, body temperature and subsequent compensatory sleep responses was examined in the rat. Animals entrained to a light-dark cycle were treated at 5 h after lights-on (CT-5) with 0.5, 1, 5 or 10 mg/kg i.p. (−)DS121 or methylcellulose vehicle. An additional group received 5 mg/kg i.p. (−)DS121 or vehicle 6 h after lights-off (CT-18). At CT-5, (−)DS121 dose-dependently increased wakefulness, locomotor activity and body temperature, and decreased both non-rapid eye movement sleep (NREM) and rapid eye movement sleep (REM) during the first 4 h post-treatment relative to vehicle controls. REM interference lasted up to 3 h longer than NREM. Low doses of (−)DS121 (0.5 and 1 mg/kg) produced relatively little waking that was not followed by significant compensatory sleep responses. In contrast, higher doses (5 and 10 mg/kg) produced compensatory hypersomnolence (robust increases in NREM immediately after the primary waking effect) that was proportional to the duration of drug-induced wakefulness. NREM recovery 24 h post-treatment was the same for the 5 mg/kg (65.4 ± 9.9 min) and 10 mg/kg (64.8 ± 9.3 min) doses, but was not proportional to prior wake duration. NREM displaced by drug-induced wakefulness was recovered completely by 24 h post-treatment at the 5 mg/kg dose, but only 63.5% recovered at 10 mg/kg. In contrast, equivalent wakefulness produced by sleep deprivation yielded 100% NREM recovery. At CT-18, (−)DS121 (5 mg/kg) increased wakefulness without disproportionately increasing locomotor activity, and was compensated fully by 24 h post-treatment. These data show that (−)DS121 dose-dependently increases wakefulness, which is followed by hypersomnolence that is proportional to drug-induced wake-promoting efficacy.

More selective modulation of a specific monoaminergic transmitter system can be achieved by administration of selective autoreceptor antagonists (Chesselet, 1984; Langer, 1981). For example, the DAAs (+)AJ76 and (+)UH232 enhance dopamine neurotransmission, primarily by blocking presynaptic D2 and/or D3 dopamine synthesis and/or release-modulating receptors (Gainetdinov et al., 1994;Gifford and Johnson, 1993; Rayevsky et al., 1995; Waterset al., 1993). The more recently developed DAA (−)DS121 is more active at release-controlling dopamine autoreceptors than (+)AJ76 and (+)UH232 (Sonesson et al., 1993, 1994). To date, only one study has examined the wake-promoting effects of DAAs. Svenssonet al. (1987) found that both (+)AJ76 and (+)UH232 produce dose-dependent increases in waking. However, it is not known whether the waking induced by DAAs elicits compensatory sleep responses analogous to those seen after psychostimulants (Edgar and Seidel, 1997) or sleep deprivation (Tobler and Borbely, 1986). In the present study we examined the acute effects of (−)DS121 on sleep/wake, locomotor activity and body temperature parameters and subsequent compensatory sleep responses in the rat.

Methods

Animal surgery.

Adult male Wistar rats (300–500 g at time of surgery, Charles River Laboratories, Wilmington, MA) were anesthetized with Nembutal (60 mg/kg i.p.) and surgically prepared with a cranial implant that permitted chronic EEG and EMG recording (Edgaret al., 1991). The cranial implant consisted of four stainless steel screws for EEG recording (two frontal at + 3.9 mm AP and ± 2.0 mm ML from bregma, two occipital at −6.4 mm AP and ± 5.5 ML from bregma). EMG was monitored by two Teflon-coated stainless steel wires positioned under the nuchal trapezoid muscles. All leads were soldered to a miniature connector before surgery and gas sterilized. The implant assembly was affixed to the skull with a combination of cyanoacrylate and dental acrylic. Body temperature and LMA were monitored via miniature transmitters (Minimitter, Sunriver, OR) surgically placed in the abdomen. A minimum of 3 weeks was allowed for postsurgical recovery.

Recording environment.

Rats were housed individually within specially modified Nalgene microisolator cages equipped with a commutator and filter-top riser, as described previously (Seidelet al., 1995). Each cage was located within separate ventilated compartments of a stainless steel cabinet. Animals hadad libitum access to food and water, and were kept in a 24-h (LD 12:12) light-dark cycle throughout the study using 4-watt fluorescent bulbs 5 cm from the cage (32–35 lux inside the cage). Animals were undisturbed for 3 days before and after drug treatment.

Automated monitoring.

Sleep/wake parameters were monitored using “SCORE”, a microcomputer-based sleep/wake and physiological data collection system. Details regarding the design and performance of this system are described elsewhere (Edgar et al., 1991; van Gelder et al., 1991). EEG was amplified 10,000 times (EEG bandpass, 1–30 Hz, −6 dB per octave) and digitized at 100 Hz. EMG was amplified similarly (bandpass, 10–100 Hz) and integrated (root mean square, 0–5 V output; Barrows RDI-8, Palo Alto, CA). Tb and nonspecific LMA were monitored by telemetry. Drinking activity was detected when animals contacted the watering spout. All variables were monitored continuously and simultaneously. Arousal states were classified on-line as NREM sleep, REM sleep or theta-dominated wake every 10 sec with use of EEG feature extraction and pattern-matching algorithms. The classification algorithm used individually taught EEG arousal-state templates plus EMG criteria to differentiate REM sleep from theta-dominated wakefulness, plus behavior-dependent contextual rules (e.g., if the animal was drinking, it was awake). Drinking and LMA were recorded as discrete events every 10 sec; Tb was recorded every 60 sec. LMA and Tb were detected by a telemetry receiver (Data Sciences, St. Paul, MN) located beneath the cage. Telemetry measures (LMA and Tb) were not part of the sleep-wake scoring algorithm; thus sleep-scoring and telemetry were independent measures.

Data quality was assured by frequent on-line inspection of the signals. Graphical and statistical summaries of the 3 days before and after drug treatment were inspected to verify scoring stability. Also, sleep-wake scoring was scrutinized carefully for artifact by off-line visual examination of raw EEG waveforms and the distribution of integrated EMG values.

Drug administration and study design.

(−)DS121 (courtesy of The Upjohn Company, Kalamazoo, MI) was dissolved in sterile 0.25% methylcellulose and injected i.p. in a volume of 1 ml/kg at CT-05 (5 h after lights-on). Doses given were 0.5, 1, 5 and 10 mg/kg or vehicle control. Separate groups of animals received 5 mg/kg (−)DS121 or vehicle at CT-18 (6 h after lights-off), or were subjected to sleep deprivation (see below). In all cases animals were assigned randomly to parallel treatment groups. Sample sizes (n) were between 8 and 14 per active treatment group, and n = 20 for vehicle control groups.

Sleep deprivation.

To determine whether drug-induced wakefulness produces compensatory sleep responses analogous to sleep deprivation, a separate group of rats was maintained awake for 4 h starting at CT-5 (n = 10). Sleep deprivation was achieved by introducing novel stimuli such as toys, paper and other objects into the cage, or if necessary, making gentle contact with the animal. Stimuli were only applied when animals attempted to sleep, as defined by visual observation and confirmed by real-time computer scoring. Four hours of sleep deprivation was found empirically to produce the same net amount of waking as 10 mg/kg (−)DS121. Animals were undisturbed for 30 h before and after sleep deprivation.

Data analysis and statistics.

The principle variables recorded were percent per hour of NREM, REM, total sleep time (defined as minutes per hour of NREM + REM) and WAKE, ASBL and MSBL (in minutes), AWBL and MWBL (in minutes), counts per hour of LMA, LMAI (defined as LMA counts per min of wakefulness; see Edgar et al., 1997; Edgar and Seidel, 1997), counts per hour of DRINK and average Tb deviation from the 24 h baseline mean. Sleep and wake bout length, LMA, DRINK and Tb parameters were averaged across post-treatment hours 1 to 4 (designated treatment effect) and hours 5 to 10 (designated recovery) for each treatment group, as well as for the corresponding values during the 24-h pretreatment period (designated baseline) (see tables 1 and 2). Differences between post-treatment drug group and vehicle were compared by one-way ANOVA. In the presence of a significant main effect, Dunnett’s contrasts (α = 0.05) assessed differences between the active treatment group and vehicle controls.

Because the duration of drug effects on NREM, REM, total sleep time and WAKE parameters varied with the dose of (−)DS121, it was important to block these data in specific time intervals so that compensatory sleep effects would not statistically cancel the primary waking effect of the drug. Two alternative approaches to this problem were used. One approach (“variable-interval” analysis) computed the duration of drug-induced waking (continuous number of hours in which drug-induced wakefulness exceeded base-line 24 h earlier) for each dose in individual animals. The waking effect was then averaged for each dose group (mean ± S.E.M.). A second approach (“sleep deficit” analysis) calculated group mean waking levels (relative to baseline) on an hourly basis. The number of hours in which the averagewaking level (immediately post-treatment) exceeded corresponding baseline values 24 h earlier defined the duration of the drug-induced waking effect.

Accumulated minutes of NREM, REM and WAKE were computed and plotted in hourly bins for 30 h post-treatment (see Edgar et al., 1997; Edgar and Seidel, 1997). These variables were calculated by serially adding the minutes of a given arousal state each hour post-treatment minus that during the corresponding baseline recorded 24 h earlier. NREM and REM sleep loss therefore was expressed as a negative accumulation value and appeared as a negative slope in the accumulation plots (see fig. 5); a positive slope in the plot indicates recovery sleep. Likewise, an increase in WAKE was expressed as a positive accumulation value and appeared as a positive slope on the accumulation plots, with a negative slope indicating recovery sleep. This analysis provided a quantitative graphical representation of the magnitude and time course of compensatory sleep responses. Maximum accumulated wake surplus and NREM and REM deficit induced by (−)DS121, and the steady-state level after compensatory sleep 24 h post-treatment, were compared between groups using one-way ANOVA. In the presence of a significant main effect, Dunnett’s contrasts compared each active treatment group to vehicle controls.

Results

Acute effects on sleep/wake and physiological parameters.

All analyses were performed relative to base line and contrasted with vehicle controls. Figures 1 and2 show the normal circadian oscillations in NREM, REM, LMA and Tb and the effects of a 5 mg/kg dose of (−)DS121 at CT-5 or CT-18, respectively, on these parameters. (−)DS121 (5 mg/kg) induced a decrease in NREM and REM sleep as well as an increase in LMA and Tb immediately after administration at CT-5. With the exception of REM sleep, which exhibited delayed compensatory sleep responses (see below), these parameters returned to normal circadian patterns by 12 h post-treatment. CT-18 administration of the same dose produced a decrease in NREM sleep as well as an increase in Tb, but the increase in LMA did not exceed vehicle control levels. Under baseline conditions (e.g., 24 h before treatment at CT-5), rats exhibited relatively high levels of NREM and REM sleep and low levels of LMA and Tb, commensurate with nocturnal sleep/wake behavior. Thus, treatment at CT-5 with 5 mg/kg (−)DS121 produced more robust effects on these variables than during the animals’ circadian active phase at CT-18. Also, in contrast to CT-5, these variables returned to normal circadian patterns by 6 h post-CT-18-treatment, with delayed compensatory REM sleep responses again being an exception.

NREM, REM, LMA and Tb circadian rhythms before and after treatment at CT-5 with (−)DS121 (5 mg/kg i.p.) (solid line,n = 10 or 11) or methylcellulose vehicle (dotted line, n = 20). The injection at CT-5 is denoted by the triangle on the x-axis. The four variables are plotted as mean ± S.E.M. across a 2.5-day period, and light/dark bars along the x-axis indicate lights on/off. Note that during hours 1 to 4 post-treatment NREM and REM are decreased relative to vehicle controls, and during hours 5 to 10 post-treatment these variables are increased, which indicates compensatory sleep responses. Conversely, both LMA and Tb are increased during hours 1 to 4 post-treatment followed by distinct periods of hypolocomotion and decreased body temperature during hours 5 to 10 post-treatment relative to vehicle controls.

NREM, REM, LMA and Tb circadian rhythms before and after treatment at CT-18 with (−)DS121 (5 mg/kg i.p.) (solid line,n = 13 or 14) or methylcellulose vehicle (dotted line, n = 20). The injection at CT-18 is denoted by a triangle on the x-axis. Data are plotted as in figure1. Note the lack of significant effects on LMA and Tb, and the delayed compensatory REM sleep response beginning approximately 12 h after treatment.

Table 1 describes the sleep/wake architecture (as reflected in sleep and wake bout lengths), LMA, LMAI, DRINK and Tb parameters during: 1) 4 h baseline (obtained during the corresponding 4-h time block 24 h before treatment), 2) treatment effect (4 h post-treatment; CT-5 to CT-9) and 3) recovery (5–10 h post-treatment; CT-9 to CT-14). These variables are also reported for the CT-18 treatment group in table2. When given at CT-5, both the 0.5 and 1 mg/kg doses of (−)DS121 were without effect on sleep/wake bout length, LMA, LMAI, DRINK and Tb parameters during the treatment effect period. In a dose-related manner, (−)DS121 (5 and 10 mg/kg) given at CT-5 significantly reduced sleep bout length (ASBL and MSBL) while increasing wake bout length (AWBL and MWBL), LMA, LMAI and Tb during the treatment effect period (table 1). DRINK was increased during the treatment effect period after treatment at the 5 mg/kg dose only. When given at CT-18, 5 mg/kg (−)DS121 also significantly reduced sleep bout length (ASBL and MSBL) while increasing wake bout length (AWBL and MWBL) and Tb during the treatment effect period, but the increase in LMA, LMAI and DRINK did not significantly exceed vehicle controls (table 2).

Under baseline conditions at CT-5, wakefulness, NREM sleep and REM sleep were not significantly different between treatment groups. (−)DS121 at 1, 5 and 10 mg/kg significantly and dose-dependently increased wakefulness (132.4 ± 1.0, 169.2 ± 6.0 and 205.6 ± 4.8 min, respectively) relative to vehicle (100.8 ± 2.4 min; ANOVA F(4,62) = 77.27, P < .001) during the 4-h treatment effect period. It should be noted, however, that lower doses (0.5 and 1 mg/kg) produced effects of shorter duration (i.e., 1–2 h) that were underestimated when calculated during this 4-h period. To assess the net wake-promoting effects of (−)DS121 at each dose more accurately, cumulative wake, NREM and REM sleep profiles were calculated (see below).

The rapid onset of action and dose-dependent efficacy of (−)DS121 is illustrated in detailed pharmacodynamic plots shown in figure3. At CT-5, 5 and 10 mg/kg (−)DS121 sustained almost 100% wake per unit time for 2 h post-treatment, with waking levels decaying rapidly thereafter. A similar pharmacodynamic profile was observed in rats treated with 5 mg/kg (−)DS121 at CT-18. The increased wake continuity produced by (−)DS121 is illustrated further in a time series plot of MWBL shown in figure4A. Before treatment at CT-5, a circadian rhythm in MWBL is evidenced, with the longest bouts occurring during the animals’ activity phase (lights-off). After treatment with 10 mg/kg (−)DS121, MWBL increased approximately 200% relative to vehicle. Five hours after treatment, MWBL returned to its usual circadian pattern.

Wakefulness induced by (−)DS121 resulted in immediate compensatory sleep responses that were proportional to the duration of drug-induced wakefulness. In figure 1, this immediate compensatory sleep response was evidenced as a robust increase in NREM sleep that exceeded normal circadian levels during the 5- to 10-h post-CT-5-treatment interval. Compensatory sleep also was evidenced by a marked increase in REM sleep relative to vehicle-treated animals in the 8- to 12-h interval after treatment at CT-5 (fig. 1) and the 12- to 18-h interval after treatment at CT-18 (fig. 2).

Compensatory sleep responses to (−)DS121 treatments at CT-5 were evidenced further by dose-dependent increases in ASBL and MSBL during the 5- to 10-h post-treatment period (table 1 and fig. 4B), and were reflected indirectly in concomitant decreases in LMA, MWBL and Tb (table 1). At lower doses (i.e., ≤1 mg/kg), small but significant increases in wakefulness did not elicit significant compensatory responses in ASBL or MSBL (table 1). The independence of sleep bouts and wake bouts is illustrated by the finding that whereas compensatory increases in ASBL and MSBL were observed during the recovery period after 5 mg/kg (−)DS121 treatment at CT-18, AWBL and MWBL remained slightly (but significantly) longer during this interval (table 2). A small decrease in Tb also was observed during this recovery period (table 2).

Cumulative changes in NREM, REM and WAKE.

Dose-dependent comparison of long-term compensatory sleep responses after drug-induced wakefulness is complicated by the fact that both the magnitude (i.e., minutes per hour of waking) and the duration of drug action (i.e., total time the drug increases waking) can vary independently with dose. The pharmacodynamic profile of drug-induced waking and the course of subsequent recovery sleep can be quantified precisely for each treatment, however, by plotting a running sum of the difference from the corresponding hour during baseline 24 h earlier for each hour post-treatment (“sleep deficit” analysis) (Edgar et al., 1997; Edgar and Seidel, 1997). Figure5 shows the group mean arousal state accumulation profiles for NREM, REM and WAKE after 5 and 10 mg/kg (−)DS121 and methylcellulose vehicle administered at CT-5. Arousal state accumulation profiles for vehicle and 5 mg/kg treatment at CT-18 are shown in figure 6. The greatest amount of drug-induced sleep loss (e.g., negative-most value for cumulated NREM or REM) or accumulated wake, the time to reach such “peak” values (computed from the group mean profiles in fig. 5), and cumulative measures of each arousal state 24-h post-treatment, are shown in table 3. When given at CT-5, (−)DS121 rapidly reduced NREM and REM sleep (evidenced by the negative slopes for NREM and REM during the first 4 h in fig. 5), but the time course of these effects differed as a function of the variable measured and as a function of drug dose. For example, 6 and 7 h after treatment with 5 and 10 mg/kg of (−)DS121, the maximum deficit was reached after 3 and 4 h for NREM but after 6 and 7 h for REM, respectively. After treatment with either higher dose, there was an intense NREM compensatory sleep response (positive slope in the NREM accumulation plots) that preceded compensatory REM sleep (see also fig.1 for comparison). Recovery of 50% of REM sleep lost to drug-induced waking was observed 6 to 7 h after recovery of 50% of NREM sleep lost (see fig. 5). Waking induced by vehicle treatment also showed NREM recovery preceding REM recovery, although the absolute magnitude of this effect is small. By 24 h post-treatment, animals had recovered approximately 100% of NREM and 65% of REM suppressed by 5 mg/kg of (−)DS121 at CT-5. In contrast, animals recovered only 65% of both NREM and REM 24 h after 10 mg/kg at CT-5.

(−)DS121 effect on cumulated changes in NREM, REM and WAKE time (based on individual values of maximum wake-promoting duration)

When administered at CT-18, the 5 mg/kg dose of (−)DS121 also suppressed NREM and REM sleep. Based on the group mean plots in figure6, peak suppression of NREM was observed 3 h after treatment (as at CT-5) and was 100% recovered within 24 h (see table 3). In contrast to treatment effects at CT-5, however, REM sleep suppression, albeit smaller in relative magnitude, was sustained much longer at CT-18 (12 h).

Although group mean accumulation profiles give a good general sense of the initial and delayed course of compensatory sleep, individual variation in the duration of the initial wake-promoting effect, and thus the onset of compensatory sleep responses, could produce underestimates of these measures (because of the smoothing effect inherent in averaging) and confound dose-dependent comparisons. Therefore, we further analyzed the data by first constructing arousal state accumulation waveforms for each individual rat in the CT-5 treatment group. The peak cumulative wakefulness, duration to reach peak wake-promoting effect, and minutes of compensatory NREM sleep in a 4-h block after peak waking then were calculated for each animal and averaged. These results are shown as three-dimensional plots in figure7; dose 0 mg/kg = vehicle. When data were analyzed in this manner, the duration of the (−)DS121 wake-promoting effect, the peak accumulated wakefulness and the initial compensatory NREM sleep response were all dose-related. Mean ± S.E.M. duration of wake-promoting effects for vehicle, 0.5, 1.0, 5.0 and 10 mg/kg were: 81.0 ± 7.8, 90.0 ± 13.2, 150.0 ± 45.6, 229.2 ± 19.2 and 267.6 ± 16.2 min, respectively. Corresponding minutes of cumulated waking were: 22.3 ± 2.6, 32.8 ± 7.4, 53.2 ± 12.6, 89.5 ± 7.7 and 135.8 ± 9.0 min, respectively. Cumulated NREM in the 4-h post-peak waking effect corresponding to the doses and wake-promoting effects above were: 13.3 ± 1.8, 10.2 ± 3.8, 18.8 ± 2.4, 34.2 ± 5.0 and 52.3 ± 2.9 min, respectively.

Total cumulated change in minutes of NREM relative to baseline 24 h earlier after treatment at CT-5 with 10 mg/kg (−)DS121 (▴, n = 13) or 4 h sleep deprivation (□, n = 10). Data are plotted as in figure 5. Note the recovery of virtually all sleep lost in the sleep deprivation group, and only partial recovery in the (−)DS121-treated group.

Discussion

Dopaminergics exert a powerful influence on the arousal state by their actions on monoaminergic projections in the brain (Holman, 1994;Ongini and Longo, 1989). Psychostimulants such as methamphetamine and d-amphetamine increase dopaminergic and noradrenergic transmission by facilitating neurotransmitter release, inhibiting neurotransmitter reuptake and inhibiting the degradative enzyme monoamine oxidase (Cho, 1993; Holman, 1994; Kuczenski, 1983). However, the wakefulness-promoting effects of psychomotor stimulants are thought to be associated primarily with dopaminergic rather than noradrenergic neurotransmission (Nishino and Mignot, 1997). These compounds also elicit strong compensatory sleep responses that are proportional to the amount of drug-induced waking (Edgar and Seidel, 1997). In the present study, (−)DS121, a substituted 3-phenylpiperidine that preferentially antagonizes presynaptic D2 and D3 dopamine autoreceptors (Sonessonet al., 1993, 1994), produced a dose-dependent increase in wakefulness and robust compensatory hypersomnolence akin to that reported for methamphetamine (Edgar and Seidel, 1997) andd-amphetamine (Touret et al., 1995), consistent with our working hypothesis that dopaminomimetic-induced wakefulness engages homeostatic compensatory sleep responses.

Treatment effects at CT-5 vs. CT-18.

The threshold wake-promoting efficacy for (−)DS121 at CT-5 was 1 mg/kg, at which dose all variables except wake time (i.e., sleep and wake bout lengths, LMA, Tb) were not affected significantly. Thus, EEG wakefulness can be a very sensitive measure of dopaminomimetic drug action. At higher doses (5 and 10 mg/kg), (−)DS121 at CT-5 dose-relatedly increased both wake bout parameters (AWBL and MWBL), which indicates that this compound not only increased wakefulness, but also consolidated wakefulness. A comparison of 5 mg/kg doses at CT-5 and CT-18 revealed that (−)DS121 can potently suppress NREM and REM sleep at both times of day. Indeed, the pharmacodynamic profile of (−)DS121 (i.e., percent wake per hour post-treatment) was very similar at each CT, which suggests that there was little interaction between drug efficacy and the circadian time-keeping system. However, the total wake time induced at CT-18 was less than at CT-5. As a nocturnal species, rats are typically awake approximately 70% time during the lights-off phase of the circadian cycle (Edgaret al., 1991; Edgar, 1994, 1996). Drug “ceiling effects” on sleep-wakefulness therefore limit the net increase in wakefulness as a function of time of day. Similar issues come to bear when contrasting LMA and Tb as a function of treatment time of day. Large increases in LMA and Tb were seen in response to 5 mg/kg (−)DS121 at CT-5. At CT-18, similar levels of LMA and Tb were observed, but were not markedly different from the usual levels (e.g., as in vehicle group or during baseline 24 h earlier). This circadian variation in LMA response to treatment is consistent with previous reports of circadian variation in locomotor responses to psychostimulants and other dopaminomimetics (Gaytan et al., 1996; Martin-Iverson and Iversen, 1989; Nagayama et al., 1978; Urba-Holmgren et al., 1977), as well as circadian variations in dopamine receptor number (Kafka et al., 1983;Wirz-Justice, 1984).

Behavioral hyperactivity.

The wake-promoting effects of low-dose (−)DS121 (1 mg/kg) at CT-5 or a moderate dose (5 mg/kg) at CT-18 were not accompanied by significant increases in LMA or LMAI relative to vehicle controls. These observations are consistent with a preliminary report that low-dose treatment with the selective dopamine reuptake inhibitor GBR 12909 increases waking without producing disproportionate motor activity (Edgar et al., 1995b). In contrast, classic psychostimulants produce hyperactivity at all doses that also produce waking (Edgar and Seidel, 1997; Wise, 1988). Thus, it seems that modest enhancement of dopamine transmission can promote wakefulness without extrapyramidal motor side effects.

The wakefulness observed after higher doses (i.e., 5 and 10 mg/kg) of (−)DS121 at CT-5 was accompanied by unambiguous behavioral hyperactivity as indexed by increases in LMA and LMAI. Other DAAs such as (+)AJ76 and (+)UH232 also have been reported to increase LMA at higher doses (Svensson et al., 1986, 1987), which is most likely because of their release-enhancing properties of dopamine in the striatum (Gainetdinov et al., 1994; Gifford and Johnson, 1993; Rayevsky et al., 1995; Waters et al., 1993). In contrast, the wake-promoting effects of dopaminomimetics are thought to be mediated by regions in the basal forebrain or brainstem (Gillin et al., 1978; Jones, 1994; Lin et al., 1996). At 10 mg/kg (−)DS121, LMAI remained elevated several hours after the primary wake-promoting effect subsided (e.g., into the recovery period). Thus, drug effects on LMA were evident in waking episodes during what was otherwise defined as the recovery sleep interval (5–10 h post-treatment interval). Although these effects seem paradoxical, the polyphasic nature of rodent sleep may account for this observation. For example, homeostatic sleep drive (e.g., sleep tendency) resulting from the drug’s initial wake-promoting and wake-consolidating action would be predicted to increase the amount and duration of subsequent sleep bouts, especially as the wake-promoting effect of the drug subsides. Such responses would be analogous to the effects of sleep deprivation, in which compensatory sleep responses are proportional to the duration of prior waking (Dijk et al., 1990; Endo et al., 1997; Tobler and Borbely, 1986). But with high drug doses, residual drug levels still could have been sufficient to increase motor activity during intermittent episodes of wakefulness during the recovery period several hours after treatment. Because hyperactivity can be manifest only during wakefulness, a compensatory sleep response can functionally gate the expression of residual drug action on locomotor activity.

Duration of drug action: cohort vs. individual effects.

Dose comparisons of compensatory sleep responses in stimulant-treated rats are inherently complicated because low-dose effects of stimulants are of shorter duration than higher doses; thus fixed interval analysis can be problematic. In the present study we used two alternative approaches to estimate the duration of the primary wake-promoting effect of (−)DS121, and in turn, to detect compensatory sleep responses in the hours after the initial waking effect peaked. Each approach revealed strengths and weaknesses. In the first approach (“variable-interval” analysis), mean arousal state accumulation profiles (see “Methods”) were used to define the duration of initial drug action on WAKE, NREM and REM, and the beginning of the respective arousal state recovery periods. This technique yielded an average pharmacodynamic profile of drug interaction with clear compensatory sleep, including estimates of net recovery sleep many hours after treatment. With this approach the net wake-promoting effect was dose-related; however, the duration of wake-promoting action of (−)DS121 only was not precisely dose-related (seetmax in table 3). In contrast, when the duration of wake-promoting action was determined first for individual animals (defined as the initial continuous duration that wakefulness exceeded the corresponding baseline 24 h earlier, computed in hourly blocks) and then averaged, there was a straightforward relation between dose, duration of wake-promoting action and increase in accumulated wakefulness (see fig. 7). The difference in results from these two approaches is likely caused by the “cohort effect” of the former technique; that is, averaging inherently smoothes data. In particular, when wake-duration estimates are computed by the group mean wake accumulation profile, the variance in individual animals makes it probable that drug-induced waking in some animals overlaps and is averaged together with compensatory sleep responses in other animals. Unfortunately, drug duration estimates from individual rats can be methodologically difficult because the individual accumulation profiles are not as smooth as the group mean profile, making it difficult to define objectively the true maxima/minima in some individual profiles. This latter approach is complicated further at threshold doses, wherein some animals respond and others do not. In the present study, nonresponders were not excluded from the analysis.

Indices of sleep homeostasis.

Relatively few studies have analyzed the compensatory sleep responses after stimulant-induced waking. Homeostatic sleep responses can be divided into two phases: an initial recovery phase usually characterized by intense hypersomnolence (i.e., increases in NREM sleep time and sleep bout lengths) such as immediately follows sleep deprivation, and a less intense but elevated level in manifest sleep tendency that may last several hours thereafter. Although some laboratories have focused on spectral correlates of EEG activity during compensatory sleep as an index of sleep homeostasis (Borbely, 1994; Borbely and Neuhaus, 1979; Dijket al., 1987, 1990), the present study used NREM bout lengths and NREM sleep time as principal markers of the compensatory sleep responses. In rats, NREM bout lengths correlate closely with EEG delta power in NREM episodes after sleep deprivation (Edgar, 1996). It is our experience that some drugs can induce subtle shifts in EEG frequency that alter EEG power assessments without altering NREM sleep bout lengths. This phenomenon was noted previously in the assessment of benzodiazepine hypnotic action, in which drugs like triazolam can increase nocturnal sleep efficiency (and therefore improve subsequent daytime alertness), yet significantly reduce EEG delta power in NREM sleep (Johnson et al., 1983). Thus, EEG delta power can be a less reliable index of compensatory sleep than measures of sleep continuity, the latter of which strongly correlates with sleep quality (Roehrs et al., 1994).

Another aspect of sleep homeostasis is reflected in the time course of NREM and REM recovery sleep. REM recovery after (−)DS121 clearly was delayed compared with NREM. This phenomenon is not unique to pharmacologically induced wakefulness. In humans, compensatory sleep responses to sleep deprivation are characterized by an initial increase in slow-wave sleep (sleep stages 3 and 4), followed by increases in REM sleep in the latter third of the night (Bonnet, 1994; Borbely, 1994;Carskadon and Dement, 1979; Kales et al., 1970). It is not clear whether REM sleep after sleep deprivation or drug-induced wakefulness is displaced by mechanisms promoting NREM sleep, by circadian time-keeping effects or by some other interaction. Because dopaminergic agonists suppress REM sleep (Radulovacki et al., 1979, 1981a, b; Trampus et al., 1991), it is possible that residual effects of the drug may inhibit NREM and REM sleep differentially. Benington and Heller (1994) have hypothesized that REM sleep propensity is a function of prior NREM duration. In the present study, compensatory REM sleep responses after (−)DS121 were markedly delayed relative to the NREM hypersomnolence. At CT-18, compensatory REM sleep was evident in the last half of the lights-on phase after drug treatment and was not associated with any significant NREM hypersomnolence.

The relatively small amount of waking induced by a low (1 mg/kg) dose of (−)DS121 was not accompanied by compensatory responses in any parameter measured. This may be because the amount of wakefulness induced was markedly different from the levels that are usually expressed in spontaneous behavior. Higher doses, however, produced both initial and delayed compensatory sleep responses, the latter of which were visualized best by NREM sleep accumulation profiles. To better understand the net recovery of NREM sleep, we contrasted a dose of (−)DS121 (10 mg/kg) that produced an amount of waking very similar to 4 h of sleep deprivation by gentle handling. Although both procedures produced comparable intense hypersomnolence 3 h after the drug-induced wakefulness or sleep deprivation, the latent recovery of NREM sleep in these two groups was very different. NREM sleep after sleep deprivation was completely (i.e., 100%) recovered by 24 h post-treatment. The higher dose (10 mg/kg) of (−) DS121 produced waking that was recovered only partially (i.e., 65%) by 24 h post-treatment. Thus, there may be two distinct phases in the compensatory sleep response with potentially different neurochemical mechanisms.

In conclusion, augmenting dopamine release with the dopamine autoreceptor antagonist (−)DS121 dose-dependently increased waking that was followed immediately by a robust compensatory sleep response. This initial hypersomnolence was characterized by increased NREM and REM sleep time and bout lengths. Twenty-four hours after treatment, NREM recovery showed a paradoxical relationship with dose- and drug-induced waking. NREM suppressed by the higher dose of (−)DS121 was recovered only partially after 24 h, whereas the same amount of NREM suppression imposed by nonpharmacologically induced sleep deprivation was 100% recovered in 24 h. Although systemic drug administration studies necessarily limit conclusions regarding dopamine as a specific modulator of ascending cortical-activating projections or the specific brain site(s) where these effects are mediated, it is important to note that many aminergic compounds that increase noradrenergic and serotonergic transmission, but lack direct effects on dopaminergic terminals (such as clonidine, fenfluramine and tricyclic antidepressants), tend to be sedating (reviewed in Obermeyer and Benca, 1996). The non-aminergic stimulant caffeine (an A1 and A2 adenosine antagonist) potently promotes wakefulness in the rat; however, subsequent compensatory sleep responses appear to be attenuated (Edgaret al., 1995a; Seidel et al., 1994) similar to that observed after 10 mg/kg (−)DS121. Adenosine receptors have been implicated as a potential mediator of physiological sleep tendency through inhibitory action on cholinergic neurons in the basal forebrain and the pontine tegmentum (Porkka-Heiskanen et al., 1997;Rainnie et al., 1994). Presently it is not known if DS-121 or related dopamine release directly interacts with the adenosine system to alter sleep-wake regulation.

Acknowledgments

The authors thank Michael Halaas, Humberto Garcia and Laura Alexandre for expert technical assistance, and Dr. William C. Dement for supporting our on-going basic sleep research program at Stanford University.